PROJECT SUMMARY/ABSTRACT Modulating the functions of key players in protein homeostasis (proteostasis) could lead to therapies for diseases from cancer to neurodegeneration. Valosin Containing Protein (VCP, p97), a member of the AAA+ (ATPases associated with various cellular activities) family of enzymes, is one of the cell?s central regulators of proteostasis. Functions as diverse as unfolding of ubiquitinated proteins from organelles, segregation of ubiquitinated proteins from protein complexes, and remodeling of organelle membranes have been ascribed to VCP. These functions are coordinated by a set of ?adaptor? proteins and ubiquitin-processing enzymes that bind to VCP. Despite the importance of these protein-protein interactions (PPI) to regulated proteostasis, there are major gaps in our understanding of how adaptor proteins link VCP?s ATPase activity to diverse cellular functions. Furthermore, point mutations in VCP cause a fatal, degenerative disease called Multisystem Proteinopathy 1 (MSP1). MSP1 is associated with multiple alterations in proteostasis that include both increased degradation of some proteins and loss of degradation of others. VCP undergoes large conformational changes during ATP hydrolysis, and MSP1 mutations alter VCPs conformational propensity. We and others have shown that PPI are also linked to VCP conformation, leading to the hypothesis that VCP?s PPI network is modulated by ATPase-dependent conformational dynamics, and that MSP1 mutations lead to dysregulation of the PPI network by altering these dynamics. MSP1 disease should therefore be viewed as a disease of network dysregulation. To address this hypothesis, we need new tools that address how adaptors bind to VCP conformations and alter ATPase activity, and how conformational-dependent binding affects the cellular activities of the VCP network. We will address these gaps through three Specific Aims. 1) We have shown that adaptor proteins sense VCP conformation. We will extend these observations to at least eight adaptor/VCP complexes and will also evaluate the effect of adaptors on ATPase activity and conformation. This analysis will provide predictions for which adaptor- dependent functions are increased or inhibited in MSP1 cells. 2) We have utilized a site-directed small-molecule discovery approach called disulfide-trapping to identify compounds that lock VCP into specific conformations. We hypothesize that inhibiting VCP dynamics will stabilize some PPI, but will inhibit biochemical and cellular functions that rely on VCP conformational dynamics. 3) We have developed phage-displayed libraries of the N- domain of VCP to select mutants that bind with high affinity and selectivity to single adaptor proteins. We hypothesize that blocking individual adaptor/VCP complexes in cells will lead to changes in VCP-mediated pathways and the ubiquitin proteome (ubiquitinome). Combining PPI measurements, small-molecule conformational locks, and protein-based PPI inhibitors will allow us to predict which VCP pathways lead to (or mitigate) MSP1 phenotypes, and which should be harnessed to develop cancer-specific VCP inhibitors.